1. Field of the Invention
The present invention is broadly concerned with novel inocula for administration to cattle or sheep in order to lessen or prevent the incidence of liver abscesses and/or foot rot. More particularly, the invention pertains to such inocula, methods of preparing the same and methods of lessening the incidence of liver abscesses and/or foot rot in cattle or sheep via administration of the inocula. In one aspect, the invention relates to an A. pyogenes-derived inoculum, and in another aspect to a multivalent inoculum comprising a first bacterial component in the form of an inactivated cell culture product of A. pyogenes and a second bacterial component in the form of an inactivated cell culture product of F. necrophorum.
2. Description of the Prior Art
Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States. This estimate is based primarily on condemnation of liver and other organs, and does not include economic losses stemming from reduced feed efficiencies and lowered weight gains. A number of studies have confirmed that cattle with abscessed livers gain less (average 4-5%) and have reduced feed efficiencies (average 7%) compared with cattle having healthy livers. The average incidence of abscessed liver in grain-fed cattle approximates 25-30%.
Liver abscesses in cattle are part of a disease complex where the abscessation is secondary to primary foci of infection in the rumen epithelium. The pathogenesis can be summarized as follows: (1) ruminal lesions are induced by acidosis that follows rapid change in diet from high-roughage to high grain, prolonged feeding of high grain diet (sometimes referred to as an all-concentrate diet), or occasionally by foreign body penetration of the rumen epithelium; (2) bacteria present in the rumen invade the epithelium and form focal abscesses in the rumen wall; and (3) bacteria enter the portal circulation, and are carried to the liver where they localize in the parenchyma with subsequent abscess formation.
F. necrophorum is the primary etiologic agent of liver abscesses in ruminant animals. The organism has been recognized as an animal and human pathogen since the late 1800s, and is associated with numerous necrotic disease conditions in domestic and wild animals. In addition to liver abscesses, the organism is also the primary etiologic agent of foot rot, foot abscesses, calf diphtheria, and is frequently isolated from cases of mastitis, metritis, and necrotic lesions of the oral cavity.
The ability of F. necrophorum to establish in the liver is attributed to the production of a toxin called leukotoxin (or leucocidin). The toxin is soluble, proteinaceous and has specificity for bovine leukocytes. The leukotoxin is believed to aid in the establishment of F. necrophorum in the liver by directly impairing the normal defense mechanism and indirectly by the damage caused by cytolytic products released from neutrophils and macrophages to the hepatic cells. Therefore, the leukotoxin elaborated from F. necrophorum plays a critical role in F. necrophorum infection of the liver.
F. necrophorum is a gram-negative, nonsporeforming, nonmotile, strictly anaerobic and pleomorphic organism. Morphologically, the organism varies from short rods to filamentous with pointed and rounded ends. Cell lengths range from coracoid bodies of 0.5-0.7 .mu.m in diameter to filaments over 100 .mu.m. Surface colonies are 1-2 mm in diameter, circular, transparent to opaque, and with some strains producing .alpha. or .beta. hemolysis. The organism ferments glucose, fructose and maltose only weakly with final pH around 5.0-6.3. It ferments lactate to acetate, propionate, and butyrate. Butyrate is the major product from lactate fermentation. Indole is produced from peptone. F. necrophorum has been isolated from the normal flora in the oral cavity, gastrointestinal cavity, and genitourinary tract of humans and animals. The organism is also known to survive in the soil.
Four biotypes (A, B, AB and C) of F. necrophorum have been described. Biotype A, most frequently isolated from liver abscesses, is more pathogenic than biotype B, which predominates in ruminal wall abscesses. Biotype AB is rarely isolated, and has pathogenicity intermediate that of biotypes A and B. Biotype C is non-pathogenic.
It has been suggested in the past to utilize F. necrophorum bacterin as an agent for immunizing cattle and sheep against liver necrosis, EPO Application No. 460480 of Dec. 11, 1991. Specifically, virulent F. necrophorum isolates are inactivated using .beta.-propiolactone, followed by addition of adjuvants. In addition, Abe et al. (Infection and Immunity, 13:1473-1478, 1976) grew F. necrophorum for 48 hours. Cells were obtained by centrifuging, washing three times with saline, and were inactivated with formalin (0.4% in saline). The inactivated cells were then injected into mice to induce immunity. Two weeks after the last booster injection, each mouse was challenged with viable cells of F. necrophorum. The mice immunized with killed cells and challenged with live cells had no detectable bacteria in the liver, lung or spleen for up to 28 days. It was concluded that immunization of mice with formalin-killed F. necrophorum conferred protection against infection. Garcia et al. (Canadian J. Comp. Med, 38:222-226, 1974) conducted field trials to evaluate the efficacy of alum-precipitated toxoids of F. necrophorum. The vaccine preparation consisted of washed cells (unlikely to contain leukotoxin) that were ruptured by sonication. The most promising result was achieved with the injection of 15.5 mg protein of cytoplasmic toxoid. In this group, the incidents of liver abscesses was reduced to 10% from an average 35% in the control group. Finally, Emery et al., (Vet. Microbiol., 12:255-268, 1986) prepared material by gel filtration of 18-hour culture supernate of F. necrophorum. This elicited significant immunity against challenged from viable F. necrophorum. The injected preparation contained endotoxin and the majority of the leukotoxic activity.
PCT Publication WO 94/00556 published Jan. 6, 1994 describes novel F. necrophorum leukotoxid vaccines as well as methods of enhancing the elaboration of leukotoxin from F. necrophorum and of producing a leukotoxin vaccine. In particular, this publication discloses that F. necrophorum bacteria are advantageously cultured in growth media at a temperature of from about 35.degree.-41.degree. C. and a pH of from about 6.5-8 for a period of from about 4-10 hours in order to maximize the production of leukotoxin. Thereafter, culturing is terminated and the leukotoxin-bearing supernate is separated and inactivated to form a vaccine.
Actinomyces pyogenes is a gram-positive, cocco-bacillary shaped, facultative organism that is associated with a number of pyogenic conditions (termed "Pyobacillosis") in animals and humans. It is frequently isolated in mixed culture with other bacteria including F. necrophorum. In liver abscesses and foot rot in cattle, A. pyogenes is the second most frequent pathogen isolated. In addition to liver abscesses and foot rot, the organism is also a frequent isolated from pyogenic infections of a number of organs such as lungs, mammary glands, joints and the uterus. The pathogenic mechanism of A. pyogenes is not well understood. Some of the factors that contribute to pathogenicity include exotoxins (hemolysin or leukotoxin) and enzymes such as proteinase, DNases, and neuraminidase. There is also evidence that A. pyogenes in cattle synergistically interacts with other bacteria including F. necrophorum and the combination may be more virulent than individual species. However, no A. pyogenes-derived vaccines or inocula for immunizing cattle and sheep against liver abscesses and/or foot rot have been prepared in the past.